Microorganism having ability to convert sterol into androst-4-ene-3, 17-dione/androsta-1,4-diene-3, 17-dione and preparation method and use thereof

ABSTRACT

Disclosed is a microorganism having an excellent ability to convert sterol into and rost-4-ene-3,17-dione (AD) and androsta-1,4-diene-3,17-dione (ADD), a method for preparation of the microorganism and use thereof, and more particularly, a mutant strain of  Mycobacterium fortuitum  ATCC 29472,  Mycobacterium fortuitum  EUG-119, a method for preparation of the mutant and use thereof in preparing AD and ADD. The mutant of the present invention has an excellent ability to convent sterol into AD and ADD which are steroid hormone precursors, compared with the known microorganisms and accordingly, is very useful for mass production of steroid hormones.

TECHNICAL FIELD

The present invention relates to a microorganism, Mycobacteriumfortuitum EUG-119, having an excellent ability to convert sterol intoandrost-4-ene-3,17-dione and androsta-1,4-diene-3,17-dione, a method forpreparation of the microorganism and use thereof, and more particularly,to a microorganism having four or more times higher conversion rate thanthat of previously known microorganisms, a method for preparation of themicroorganism and use thereof in preparing androst-4-ene-3,17-dione andandrosta-1,4-diene-3,17-dione.

PRIOR ART

Steroids, which are secretary hormones released from the adrenal cortex,the testicle, the ovary or the placenta, the corpus luteum aresynthesized from cholesterol. They are classified into about fiveclasses according to their physiological activities, as follows: sexhormones of androgen (androsterone, testosterone, etc.) and estrogen(estradiol, etc.) playing a critical role in the development ofsecondary sex characteristics in men and women, respectively, gestogen(progesterone, etc.) stimulating and maintaining pregnancy,glucocorticoid (cortisone, hydrocortisone, etc.) stimulatinggluconeogenesis and increasing liver glycogen levels by catabolism ofproteins, and mineralcorticoid (deoxycorticosterone, aldosterone, etc.)playing a important role in maintaining the balance of electrolytes andwater in a body.

Levels of the above hormones become unbalanced in a body owing toincreased stresses and exposure to environmental hormones in accordancewith the advance of civilization, resulting in occurrence of manydiseases, and widespread use of the steroid hormones is conducted fortherapy of the diseases. In particular, synthesized estrogens areessentially used in artificial fertilization and therapy of sterilepatients, and glucocorticoids play a role in relieving the pain causedby various inflammations, such as iriditis, arthritis and the like. Inaddition, Addison's disease, which is fatal, can be treated byadministration of deoxycorticosterone and hydrocortisone.

There have been conducted a variety of researches into in vitrosynthesis of steroid hormones to meet the increased demands as describedabove, and one of them relates to production of steroid hormoneprecursors using a microorganism. Mamoli and Vercellone (Ber. 70470 andBer. 702079, 1937) reported reduction of 17-ketosteroids to17-β-hydroxysteroid by fermentation of yeast. Also, Peterson and Murrayin U.S. Pat. No. 2,602,769 disclosed a method for producing11α-hydroxylation of progesterone using the fungus of genus Rhizopus,and Kraychy in U.S. Pat. No. 3,684,657 disclosed a process forandrost4-ene-diene-3,17-dione, androsta-1,4-diene-3,17-dione, and20α-hydroxymethylpregna-1,4-dien-3-one from a steroid comprising a17-alkyl using Mycobacterium species.

To achieve mass production of steroid hormones, there were attempts toisolate microorganisms using sterols as a sole carbon source and tomodify structure of the sterol used as a substrate for fermentation, andalso, to enhance an acquisition rate of steroids with the use ofchemical additives capable of preventing degradation of sterol nucleus,such as a metal, a metal absorbent and a metal reducing agent, have madegreat progress (Marsheck, et. al., Applied microbiology. 23(1). 72-77,1972). Moreover, there were also performed researches for improvingproductivity of steroid precursors, in which mutagenesis was performedon microorganisms isolated from soil through physical and chemicaltreatments, and particularly, Upjohn in U.S. Pat. No. 4,293,644described a method for yield-up androst-4-ene-3,17-dione (hereinafterreferred to as AD) predominantly and a small amount ofandrosta-1,4-diene-3,17-dione (hereinafter referred to as ADD) from avariety of sterols by a mutant strain from Mycobacterium (ATCC 29472).

In accordance with increased demands for steroid hormone medicines,there is a need for mass production of the above mentioned hormoneprecursors, especially AD and ADD, which are important precursorcompounds for in vitro synthesis of steroids. However, the previouslyknown microorganisms have low productivity in synthesizing AD and ADD.With this regard, it is urgently required to develop microorganismshaving a high conversion rate of sterols into AD and ADD.

DISCLOSURE OF THE INVENTION

As a result of many trials by the inventors of the present invention, itwas found that a Mycobacterium fortuitum EUG-119 mutant strain ofMycobacterium fortuitum (ATCC 29472), has an excellent conversionefficiency of sterols into AD and ADD, and the present invention wasaccomplished on the basis of the result.

Accordingly, it is an object of the present invention to provide amutant microorganism having an excellent conversion efficiency ofsterols into AD and ADD.

It is another object of the present invention to provide a method forpreparing a mutant microorganism having an excellent conversionefficiency of sterols into AD and ADD.

It is still another object of the present invention to provide a methodfor preparing AD and ADD using a mutant microorganism having anexcellent conversion efficiency of sterols into AD and ADD.

To achieve the above objects, the present invention provides a methodfor preparing Mycobacterium fortuitum EUG-119, deposited in the KoreanCulture Center of Microorganisms (KCCM) with accession No. KCCM-10259,comprising the steps of: (a) culturing Mycobacterium fortuitum ATCC29472 strain in a sterol-containing culture medium; (b) treating thecultured Mycobacterium fortuitum with nitrosoguanidine (NTG); and (c)growing the nitrosoguanidine-treated bacteria in a medium supplementedwith sterol or AD and ADD at a concentration of 0.1-2.0 g/L, wherebymutant strains growing rapidly in a sterol-adding medium and slowly inan AD and ADD-adding medium can be selected.

A method for preparing AD and ADD of the present invention comprisessteps of culturing the mutant microorganism, Mycobacterium fortuitumEUG-119, in a sterol-containing liquid medium and recovering AD and ADDfrom the cultivated medium.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a HPLC chromatogram of AD and ADD in the cultured medium ofMycobacterium fortuitum EUG-119.

BEST MODES FOR CARRYING OUT THE INVENTION

In accordance with the present invention, there was used Mycobacteriumfortuitum (ATCC 29472), which is capable of transforming to steroidsfrom cholesterols by fermentation.

To prepare Mycobacterium fortuitum EUG-119 of the present invention, atfirst, Mycobacterium fortuitum strain (ATCC 29472) was grown to O.D. 0.6to 0.8, and then treated with nitrosoguanidine (NTG) at an amountcapable of killing 99.9% of organisms. NTG, which is a compound commonlyused in producing mutant strains of microorganisms, induces mutationsthrough substitution of GC pair with AT pair during DNA replication bymethylation at C₆ of guanine. The amount of NTG is 300 to 400 μg per 5ml of medium, and the preferred amount is 330 μg per 5 ml. In addition,the mutation can be accomplished by UV irradiation, INH (Isoniazid) andother mutagenic compounds.

From the NTG-treated strains, there were selected mutant strains havingboth a high growth rate on a sterol-adding solid medium and a low growthrate on a AD and ADD-adding solid medium, and finally, a mutant strainMycobacterium fortuitum EUG-119 having the highest conversion rate wasobtained through the flask cultivation.

Preferably, sterol or AD and ADD used in the strain selection is addedto a medium at an amount of 0.1 to 2.0 g/L, and preferably, 0.4 to 0.6g/L. Sterol can be selected from the group consisting of sitosterol,cholesterol, stigmasterol and campesterol, and it is preferable that thesterol is from cholesterol.

Mycobacterium fortuitum EUG-119 prepared by the method as describedabove was deposited in Korean Culture Center of Microorganisms withaccession No. KCCM 10259 on Apr. 14, 2001.

In an embodiment of the present invention, in which the finally selectedmutant strain, Mycobacterium fortuitum EUG-119 was incubated and itsconversion rate of cholesterol into AD and ADD was analyzed, the mutantstrain showed about 2.3 times higher conversion rate in a small scalecultivation and about 4.2 times higher in a large scale cultivation,than a wild type strain. AD and ADD were recovered at a purificationyield of about 80% from 2 L of the mutant strain-cultivated medium bycommon separation and purification processes. It was found that, 2 daysafter cultivation, the mutant strain produced higher concentrations ofAD and ADD than the wild strain.

Accordingly, Mycobacterium fortuitum EUG-119 of the present inventioncan be used for mass production of AD and ADD. Such a method comprisesthe steps of culturing Mycobacterium fortuitum EUG-119 in asterol-containing liquid medium and recovering AD and ADD from thecultivated medium.

When Mycobacterium fortuitum EUG-119 of the present invention is used inpreparing AD and ADD used as a precursor for steroid hormone synthesis,AD and ADD can be obtained at a high yield.

The present invention will be explained in more detail with reference tothe following examples in conjunction with the accompanying drawing.However, the following examples are provided only to illustrate thepresent invention, and the present invention is not limited to them.

EXAMPLE 1 Preparation of Mutant Strains

To induce mutation, Mycobacterium fortuitum strain (ATCC 29472) wasinoculated in YNG medium, and cultured upon reaching O.D. 0.6 to 0.8, 5ml of the cultured medium was centrifuged, obtaining a cell pellet. Theobtained pellet was washed twice with sterilized 0.1 M sodium citratebuffer (pH 5.6) containing 0.5% Tween 80, and resuspended in 5 ml of thebuffer, and NTG was added to the cell suspension at a concentration of330 μg/ml. NTG-treated cell suspension was incubated with shaking at 37°C. for 90 min, and then centrifuged, obtaining a cell pellet and theobtained cell pellet was washed three times with a sterilized 0.1 Msodium phosphate buffer (pH 7.0) and resuspended in the buffer. Thefinal cell suspension was loaded onto a SMI solid medium and incubatedat 37° C. for 3-4 days to form colonies.

EXAMPLE 2 Selection of Mutant Strains

Colonies obtained from Example 1 were inoculated in MS1,MS1+cholesterol, MS1+ADD (or AD) and YNG solid media and incubated at30° C. for 3 days to select a strain having both high growth rate incholesterol-containing medium (MS1+cholesterol medium) and a low growthrate in ADD (or AD)-containing medium, and the selected strain wasdesignated as Mycobacterium fortuitum EUG-119. The MS1 medium, a minimuminorganic medium, was used as a control, and compositions of the mediaare shown in Table 1.

TABLE 1 Culture medium (%) SM4 (Fermentation Ingredient YNG SM1 MS1medium) Yeast extract 0.1 — — 0.5 Nutritional broth 0.8 — — — Glycerol0.5 1 — — Tween 80 0.1 — — 0.01 NaCl — — 0.0005 — K₂HPO₄ — 0.05 0.020.04 KH₂PO₄ — — — 0.08 NH₄Cl — 0.1 — — MgSO₄7H₂O — 0.05 0.25 0.2FeCl₃6H₂O — 0.005 — — NH₄NO₃ — — 0.1 — FeSO₄7H₂O — — 0.0001 0.0005Ammonium acetate — — — 0.15 ZnSO₄7H₂O — — — 0.0002 MnCl₄4H₂O — — —0.00005 Glucose — — — 1 Agar — 1.5 1.5 — pH 7.0 — — 7.5

EXAMPLE 3 Investigation for Conversion Rate of Cholesterol into AD andADD in M. fortuitum EUG-119 Strain

Mutant strain, M. fortuitum EUG-119, selected from Example 2 waspre-cultured in 5 ml of YNG medium, and then cultivated at 30° C. for120 hours at 200 rpm in 100 ml SM4 fermentation medium (refer toTable 1) containing 1 g glucose, 0.5 g yeast extract, 0.01 g Tween 80and various inorganic salts. Cholesterol was dissolved with acetone,because it is not dissolved well in culture media, and cholesterolsuspension in acetone was added to the medium at an amount of 0.1 gcholesterol per 100 ml. After the incubation, cultured media wereextracted with ethyl ether and petroleum ether and then dissolved with2-propanol, and used in an analysis of amounts of AD and ADD produced byM. fortuitum EUG-119 strain from cholesterol, which was carried out byhigh pressure liquid chromatography (HPLC). A conversion rate ofcholesterol into AD and ADD was represented as yield calculated by aratio of molar concentration of AD and ADD and molar concentration ofadded cholesterol. Also, there were investigated produced amounts of ADand ADD and a conversion rate of wild strain, M. fortuitum (ATCC 29472).The results are shown in Table 2.

TABLE 2 Wild strain M. fortuitum EUG-119 Yield (Molar %) 27 64 Producedamounts of AD and 199 472 ADD (mg/L) Note: Yield = Molar concentrationof produced AD and ADD/Molar concentration of added cholesterol

As shown in Table 2, mutant strain, M. fortuitum EUG-119 showed about2.3 times higher conversion rate of cholesterol into AD and ADD thanwild strain, M. fortuitum ATCC 29472.

EXAMPLE 4

Mutant strain pre-cultured in 100 ml medium, prepared by the same methodas Example 3, was inoculated into a 5 L fermentor containing sterilized2.5 L medium (pH 8.0) and incubated at 1 vvm(aeration flow rate) for 120hours at 30° C. with shaking at 600 rpm. Cholesterol dissolved inacetone was added to at an amount of 5 g/L. Produced amounts of AD andADD and conversion rate of cholesterol into AD and ADD were analyzed bythe same method as Example 3, and the results are shown in Table 3.

TABLE 3 Wild strain M. fortuitum EUG-119 Yield (Molar %) 14 59 Producedamounts of AD and 510 2,177 ADD (mg/L)

As shown in Table 3, the mutant strain, M. fortuitum EUG-119, exhibitedabout 4.2 times higher conversion rate than the wild strain.

EXAMPLE 5

AD and ADD were purified from the culture broth prepared in Example 4.2.5 L of the culture broth was adjusted to pH 3.0 and centrifuged at5000 rpm for 10 min at 4° C. The resulting biomass pellet was extractedby suspension in 70% acetone and filtered, and after evaporation ofacetone, held at 5-10° C., allowing precipitation of hormones. Theresulting precipitate was filtered and dried at 55° C., and the driedprecipitate was added with hexane to remove remaining cholesterol, andthen filtered and dried again, and the resulting crude hormoneintermediates were recovered. Recovery yields for hormone intermediatesare shown in Table 4.

TABLE 4 Weight (g) of biomass Yield (%) Culture broth 5.2 100 Afteracetone extraction 4.6 88 Crude hormones 4.2 81

As shown in Table 4, purification yield of hormone intermediates wasabout 80% from the 2.5 L culture broth of the mutant strain, M.fortuitum EUG-119.

EXAMPLE 6

The hormone intermediates produced by a wild type of M. fortuitum and amutant strain, M. fortuitum EUG-119, were measured for concentrationusing HPLC. The culture broth medium containing the wild type or themutant type of M. fortuitum, was extracted twice with four volumes of amixture of diethyl ether and petroleum ether in a ratio of 1:1, and thesolvents were then evaporated. The extracted hormones were suspended inisopropyl alcohol for quantitative analysis, with resort to HPLCutilizing Pegasil ODS (4.6×250, 5 μm, 120 Å, Senshu Pak, Japan) as acolumn under conditions of a flow rate of 1.0 ml/min, 2,700 psi and 250nm, and the mobile phase was analyzed using 50% THF (refer to FIG. 1).

As a result, it was found that, from 2 days after culturing, the mutantstrain, M. fortuitum EUG-119, produced AD and ADD in higher levels thanthe wild strain, M. fortuitum ATCC 29472.

EXAMPLE 7

Mycological properties of mutant strain, Mycobacterium fortuitumEUG-119, were investigated using the known methods, and are as follows.

M. fortuitum EUG-119 can grow at 30-35° C., utilizing sugars includingglucose, polysaccharides, glycerol, fatty acids, sterols, and the likeas a carbon source. Also, M. fortuitum EUG-119, which is a Gram (+)strain, contain abundant fats in its cell wall and grows forming yellowcolonies.

As a whole, M. fortuitum EUG-119 of the present invention shows similarmorphological and physical properties to the known M. fortuitum ATCC29472 strain, but has an excellent ability to convert sterol into AD andADD by its fermentation action.

INDUSTRIAL APPLICABILITY

As described above, Mycobacterium fortuitum EUG-119 of the presentinvention has an excellent conversion efficiency of sterol to AD andADD, which can be used as hormone precursors, in comparison with thepreviously known microorganisms, especially M. fortuitum ATCC 29472.Accordingly, M. fortuitum EUG-119 of the present invention is greatlyuseful for mass production of steroid hormones.

1. The Mycobacterium fortuitum cell line deposited as KCCM-10259.
 2. Amethod for preparing Mycobacterium fortuitum KCCM-10259, comprising thesteps of: (a) culturing Mycobacterium fortuitum ATCC 29472 strain in asterol-containing culture medium to reach an optical density of 0.6-0.8;(b) treating the cultured Mycobacterium fortuitum with nitrosoguanidineat a concentration of 300-400 μg/ml; (c) growing thenitrosoguanidine-treated bacteria in a medium supplemented with sterolor androst-4-ene-3,17-dione and androsta-1,4-diene-3,17-dione at aconcentration of 0.1-2.0 g/L, whereby mutant strains growing rapidly ina sterol-adding medium and slowly in an androst-4-ene-3,17 dione andandrosta-1,4-diene-3,17-dione-adding medium can be selected; and (d)obtaining Mycobacterium fortuitum KCCM-10259.
 3. The method as set forthin claim 2, wherein the sterol is selected from the group consisting ofsitosterol, cholesterol, stigmasterol and campesterol.
 4. A method forpreparing androst-4-ene-3,17-dione and androsta-1,4-diene-3,17-dione,comprising the steps of culturing Mycobacterium fortuitum KCCM-10259 ina sterol-containing liquid medium and recoveringandrost-4-ene-3,17-dione and androsta-1,4-diene-3,17-dione from thecultivated medium.
 5. The method as set forth in claim 4 wherein thesterol is selected from the group consisting of sitosterol, cholesterol,stigmasterol and campesterol.